Figure 3.
Figure 3. Differential gene expression in cultured monocytes/macrophages stimulated with CCL18. (A) Monocytes cultured for 3 days were stimulated with 40 nM CCL18 (or PBS control) for 3.5 hours. Differential gene expression was detected using cDNA arrays from the Stanford functional genomics facility, as described in “Gene array analysis and RT-PCR.” The 100 genes of known function with the greatest differential expression were plotted on a pie chart divided into protein classes. Results of the gene array analysis are provided as supplemental material (Table S1, available on the Blood website; see the Supplemental Figures link at the top of the online article). (B) To verify gene array results, semiquantitative RT-PCR was performed with primers for SCF, IL-15, caspase-8, and actin as a household gene. Samples were prepared as described in panel A. In donors 2 and 3, LPS (100 ng/mL) was used as a known stimulus for monocytes. (C) Detection of IL-15 on cultured monocytes by FACS. Surface expression of IL-15 was detected by FACS in monocytes stimulated with CCL18 for 4 hours. Mean fluorescence channel increased from 63 in unstimulated cells to 118 in CCL18-stimulated cells. Results of 1 of 3 representative experiments are shown.

Differential gene expression in cultured monocytes/macrophages stimulated with CCL18. (A) Monocytes cultured for 3 days were stimulated with 40 nM CCL18 (or PBS control) for 3.5 hours. Differential gene expression was detected using cDNA arrays from the Stanford functional genomics facility, as described in “Gene array analysis and RT-PCR.” The 100 genes of known function with the greatest differential expression were plotted on a pie chart divided into protein classes. Results of the gene array analysis are provided as supplemental material (Table S1, available on the Blood website; see the Supplemental Figures link at the top of the online article). (B) To verify gene array results, semiquantitative RT-PCR was performed with primers for SCF, IL-15, caspase-8, and actin as a household gene. Samples were prepared as described in panel A. In donors 2 and 3, LPS (100 ng/mL) was used as a known stimulus for monocytes. (C) Detection of IL-15 on cultured monocytes by FACS. Surface expression of IL-15 was detected by FACS in monocytes stimulated with CCL18 for 4 hours. Mean fluorescence channel increased from 63 in unstimulated cells to 118 in CCL18-stimulated cells. Results of 1 of 3 representative experiments are shown.

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